α2-macroglobulin (α2M) is a member of an α-macroglobulin family of proteins found in plasma and egg whites of a broad range of animal species. α2M is present in human plasma at relatively high levels (i.e., 2-4 mg/ml) and is produced by several cell types, such as hepatocytes, lung fibroblasts, macrophages, astrocytes and tumor cells. Borth W, “Alpha 2-macroglobulin. A multifunctional binding and targeting protein with possible roles in immunity and autoimmunity,” Ann. N.Y. Acad. Sci. 737:267-272 (1994), incorporated herein by reference as if set forth in its entirety.
α2M was initially thought to function in plasma and tissue as a humoral defense barrier that binds host or foreign peptides and particles via exposed 39 amino acid “bait regions” present on each of four identical 185 kDa subunits. Each bait region contains sites at which various proteinases, or other nucleophiles, can cleave the subunits, thereby activating α2M by changing the subunit conformation, and exposing a highly reactive internal thioester. Borth W, “Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics,” FASEB J. 6:3345-3353 (1992); and Sottrup-Jensen L, “Alpha-macroglobulins: structure, shape, and mechanism of proteinase complex formation,” J. Biol. Chem. 264:11539-11542 (1989). The thioester covalently binds and entraps the proteinase or nucleophile, inhibiting further activity by steric hindrance. For example, α2M inhibits metalloproteinases belonging to a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family, see Tortorella M, et al., “Alpha 2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes,” J. Biol. Chem. 279:17554-17561 (2004), but was previously reported that it does not inhibit proteinases with astacin-like protease domains, see Baker A, et al., “Metalloproteinase inhibitors: biological actions and therapeutic opportunities,” J. Cell Sci. 115:3719-3727 (2002).
Activated α2M is also a targeting carrier for cytokines or growth factors (e.g., TGF-β, PDGF; IL-1β, basic FGF and NGF) involved in modulating biological responses of various cell types. The cytokine or growth factor dissociates either on the cell surface of a cell expressing the low-density lipoprotein receptor-related protein (LPR) or in an endocytic compartment within the cell expressing the LPR. Activated α2M binds to LRP, which results in rapid clearance of α2M-proteinase complexes from the plasma or extracellular space for subsequent catabolism. In addition, α2M functions as a protective factor against many pathogens by binding to certain peptides (e.g. toxins or cell surface proteins) of some parasites, bacteria and viruses.
Bone morphogenetic protein-1 (BMP-1) is a prototype of a subgroup of structurally similar, secreted metalloproteinases having an astacin-like protease domain, CUB protein-protein interaction domains and EGF motifs. Bond J & Benyon R, “The astacin family of metalloendopeptidases,” Protein Sci. 4:1247-1261 (1995). In mammals, members of this subgroup proteolytically cleave precursors into mature, extracellular matrix-(ECM) forming proteins. Members of this subgroup also activate some members of the TGF-β superfamily in a broad range of species by cleaving extracellular protein antagonists. Thus, mammalian BMP-1-like proteinases (i.e., BMP-1, mammalian Tolloid (mTLD) and mammalian Tolloid-like 1 and 2 (mTLL-1 and mTLL-2, respectively)) are likely to be involved in forming ECM and in signaling by certain TGF-β-like molecules in morphogenetic events and homeostasis.
BMP-1 and Tolloid-like proteinases have a distinct protein domain, structure that includes (starting at the N-terminus) a signal peptide (for secretion), a prodomain (that must be cleaved to activate the proteinase), a conserved protease domain found in the astacin M12A family of metzincin metalloproteases, and then a number of CUB and EGF-like protein-protein interaction domains. See Ge G & Greenspan D, “Developmental roles of the BMP1/TLD metalloproteinases,” Birth Defects Res. (Part C) 78:47-68 (2006); and Hopkins D, et al., “The bone morphogenetic protein 1/Tolloid-like metalloproteinases,” Matrix Biol. (Epub ahead of print, May 18, 2007), each of which is incorporated herein by reference as if set forth in its entirety. These proteinases play diverse roles in morphogenetic events, via biosynthetic cleavage of precursors into mature functional proteins involved in formation of the ECM, and via activation of certain members of the TGFβ superfamily of growth factors. Greenspan D, “Biosynthetic processing of collagen molecules,” Top. Curr. Chem. 247:149-183 (2005).
In particular, BMP-1-like proteinases (1) process type I-III procollagen C-propeptides to yield the major fibrous components of ECM; (2) cleave a zymogen to produce active lysyl oxidase enzyme that catalyzes covalent cross-linking in collagen fibers; and (3) process procollagen N-propeptides, and in some cases C-propeptides, of the minor fibrillar collagen types V and XI. Ge G, et al., “Bone morphogenetic protein-1/tolloid-related metalloproteinases process osteoglycin and enhance its ability to regulate collagen fibrillogenesis,” J. Biol. Chem. 279:41626-41633 (2004). The latter are incorporated into growing fibrils of collagen types I and II, respectively, and appear to control the geometries of the resulting heterotypic fibrils.
Because BMP-1-like proteinases provide most, if not all procollagen C-proteinase (pCP) activity in vivo, and because C-propeptide removal is essential for collagen fibrillogenesis, the BMP-1-like proteinases are attractive targets for therapeutic interventions where inhibition of collagen fibrillogenesis is desirable. Although the formation of collagen fibrils is essential to morphogenesis and to healing of wounds and bone fractures in the adult, excessive formation of fibrous collagenous ECM causes much morbidity in the general population. These conditions include keloids (excessive skin scarring), surgical adhesions, and deep-seated fibroses of organs including lungs, liver and kidneys. The deep-seated fibroses are particularly ominous, as the replacement of parenchymal tissue by scar tissue composed essentially of fibrous, collagenous ECM destroys organ function.
Accordingly, there is a need for new methods and compositions for treating fibrotic disorders, particularly those that inhibit the activities of BMP-1-like proteinases.